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European researchers have developed a technique for the simultaneous analysis of DNA ploidy (DNA content), proliferation (fraction of S-phase cells), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, TUNEL) in fresh and paraffin-embedded tissues. To obtain material from paraffin blocks for analysis, 90-µm sections were deparaffinized and rehydrated; nuclei were obtained by subtilisin digestion followed by neutralization of the protease. TUNEL staining incorporated a FITC label and DNA was stained with 4'6-diamidino-2-phenyleindole (DAPI). Analysis was performed using a double beam flow cytometer equipped with a mercury arc lamp and argon ion laser. Very similar results were obtained for embedded and fresh tissues, except that paraffin-embedded material had a larger fraction of TUNEL-negative sub-G1 events, attributed to nuclear fragments from sectioned cells. It was important for tissue to contain minimal numbers of necrotic cells, as remaining nuclei in necrotic areas were TUNEL-positive.
Heiden T, Castanos-Velez E, Andersson LC, Biberfeld P: Combined analysis of DNA ploidy, proliferation, and apoptosis in paraffin-embedded cell material by flow cytometery. Lab Invest 80:1207–1213, 2000.[Medline]
Following the initial productive infection of upper respiratory tract epithelium, bovine herpesvirus 1 (BHV-1) becomes latent in sensory neurons of the trigeminal ganglion. Latency is associated with production of the latency-related transcript (LRT) in these cells. Reactivation of latent virus can be induced experimentally by administration of dexamethasone. Investigators have now shown by polymerase chain reastion that CD4+ lymphocytes in the tonsils of latently infected calves also contain BHV-1 DNA and low levels of LRT. Beginning 6 hours after dexamethsone treatment, levels of LRT in the tonsils fell, other viral transcripts increased, and apoptosis of tonsillar lymphocytes was initiated. These findings strongly suggested that BHV-1 can establish latency in tonsillar tissue as well as in sensory ganglia.
Winkler MTC, Doster A, Jones C: Persistence and reactivation of bovine herpesvirus 1 in the tonsils of latently infected calves. J Virol 74:5337–5346, 2000.
The European Commission evaluated diagnostic tests for their suitability for rapid, large-scale bovine spongiform encephalopathy (BSE) testing. Their results were summarized in the journal Nature and are available on the Commission's web site (http://europa.eu.int/comm/dg24/health or http://www.irmm.jrc.be). Four antibody-based tests required less than 24 hours and had high throughput potential. These tests included a non-competitive immunometric technique (E.G.&G. Wallac), an immunoblotting technique (Prionics), a chemiluminescent ELISA assay (Enfer), and a sandwich immunoassay (CEA). The latter three had 100% sensitivity and 100% specificity at the pre-determined cut-off point. The sandwich immunoassay was able reliably to detect the lowest level of PrPSc.
Moynagh J, Schimmel H: Tests for BSE evaluated. Nature <400:105, 2000.
Mice carrying the urokinase-type plasminogen activator (uPA) as a transgene under the control of the major urinary protein were constructed. Becuase uPA expression was toxic to hepatocytes, the livers in these mice were gradually replaced by newly arisen hepatocytes that had deleted the transgene. During the replacement process, transplantation of mature donor hepatocytes carrying a second marker transgene allowed them to participate in liver replacement. Thus, mice were created with livers composed of approximately 65% host hepatocytes that had deleted the uPA transgene and 35% donor hepatocytes expressing a unique marker. The mice were used to determine the origin of hepatic foci formed during liver regeneration following administration of 1,4-bi[N,N'-di(ethylene)phosphamide]piperazine (dipin) and partial hepatectomy. Although such hepatic foci are generally believed to arise from liver stem cells via an oval cell intermediate, these studies showed that 20% of the foci actually arose from mature donor hepatocytes.
Braun KM, Sandgren EP: Cellular origin of regenerating parenchyma in a mouse model of severe hepatic injury. Am J Pathol 157:561–569, 2000.
Equine infectious anemia virus (EIAV) causes an initial acute illness that evolves into a chronic relapsing disease and ultimately resolves into an inapparent carrier state. In a recent study, EIAV viremia and the immune response to the virus was followed in four experimentally infected ponies for three years. These studies revealed that there was considerable variation in levels of viral replication among the ponies at all stages of disease. On the other hand, the evolution of the immune response to the virus was similar in all ponies and involved the generation of robust humoral and cellular responses. Thus, levels of viral replication were not correlated with the immune response to the virus and were not reliable indicators of viral load.
Hammond SA, Li F, McKeon BM, Cook SJ, Issel CJ, Montelaro RC: Immune responses and viral replication in longterm inapparent carrier ponies inoculated with equine infectious anemia. J Virol 74: 5968–5981, 2000.
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